Neue Strukturdaten geben Einblick in die Bewegungen von Transportproteinen in der nicht‐ribosomalen Peptidsynthese

Kittilä, Tiia; Mollo, Aurelio; Charkoudian, Louise K.; Cryle, Max J.

doi:10.1002/ange.201602614

Cited by 6 publications

(4 citation statements)

References 53 publications

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“…. The acyl and peptidyl carrier moieties in PKS and NPRS act as a type of swinging arm linker to enable facilitated transport of the intermediate (see also Figure B) …”

Section: Multienzyme Complexes Fusion Proteins and Substrate Channelingmentioning

confidence: 99%

Cascades in Compartments: En Route to Machine‐Assisted Biotechnology

et al. 2017

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Biological compartmentalization is a fundamental principle of life that allows cells to metabolize, propagate, or communicate with their environment. Much research is devoted to understanding this basic principle and to harness biomimetic compartments and catalytic cascades as tools for technological processes. This Review summarizes the current state-of-the-art of these developments, with a special emphasis on length scales, mass transport phenomena, and molecular scaffolding approaches, ranging from small cross-linkers over proteins and nucleic acids to colloids and patterned surfaces. We conclude that the future exploration and exploitation of these complex systems will largely benefit from technical solutions for the integrated, machine-assisted development and maintenance of a next generation of biotechnological processes. These goals should be achievable by implementing microfluidics, robotics, and added manufacturing techniques supplemented by theoretical simulations as well as computer-aided process modeling based on big data obtained from multiscale experimental analyses.

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“…. The acyl and peptidyl carrier moieties in PKS and NPRS act as a type of swinging arm linker to enable facilitated transport of the intermediate (see also Figure B) …”

Section: Multienzyme Complexes Fusion Proteins and Substrate Channelingmentioning

confidence: 99%

Cascades in Compartments: En Route to Machine‐Assisted Biotechnology

et al. 2017

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“…. Die Acyl‐ und Peptidylcarrier‐Domänen in PKS und NPRS fungieren als Schwingarm‐Linker, der den “erleichterten Transport” des Intermediats ermöglicht (siehe auch Abbildung B).…”

Section: Multienzymkomplexe Fusionsproteine Und Substratkanalisierungunclassified

Kaskaden in Kompartimenten: auf dem Weg zu maschinengestützter Biotechnologie

et al. 2017

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Biologische Kompartimentierung ist ein fundamentales Prinzip des Lebens, das Zellen den Stoffwechsel, die Vermehrung und die Kommunikation mit ihrer Umgebung ermöglicht. Es wird viel am Verständnis dieses grundsätzlichen Prinzips geforscht, um biomimetische Kompartimente und katalytische Kaskaden als Hilfsmittel für den technologischen Fortschritt zu nutzen. Dieser Aufsatz fasst den aktuellen Stand der Entwicklung zusammen und legt besondere Schwerpunkte auf die Größenordnung der Systeme, den Stofftransport und die Anordnung katalytischer Einheiten mithilfe von molekularen Gerüsten. Dabei reicht das Spektrum von kleinen Vernetzern über Proteine und Nucleinsäuren bis zu Kolloiden und strukturierten Oberflächen. Die Schlussfolgerung lautet, dass die zukünftige Erforschung und Anwendung dieser komplexen Systeme stark von technischen Lösungen profitieren wird, die eine integrierte, maschinengestützte Entwicklung und Durchführung einer nächsten Generation biotechnologischer Prozesse ermöglichen. Dieses Ziel sollte durch Implementierung von Mikrofluidik, Robotik und ergänzenden Fertigungsverfahren erreichbar sein und maßgeblich durch Simulationen und computergestützte Prozessmodellierung auf Basis von “Big Data” aus multiskalaren experimentellen Analysen ergänzt werden.

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“…S-methyl-thiomethanesulfonate (MTSL) spin label experiments showed no particular interactions of the PPant-arm and the T domain [66] . Behaving like a swinging arm, the covalent bound 4ʹ-PPant facilitates the pass of substrates to the adjacent reaction centres [67] . However, the helices α2, α3…”

Section: Thiolation (T) Domain / Peptidyl Carrier Protein (Pcp)mentioning

confidence: 99%

“…and their connection loop exhibit a distinct (mainly hydrophobic) interaction with the A, C and TE domain [67,68] and likewise allow a structural mechanism to guide the PCP between active sites [60] .…”

Section: Thiolation (T) Domain / Peptidyl Carrier Protein (Pcp)mentioning

confidence: 99%

Non-ribosomal peptide synthetase engineering focusing on the condensation domain and the condensation/adenylation domain interface

Kranz¹

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Non-ribosomal peptide synthetases (NRPSs) are modular biosynthetic megaenzymes producing many important natural products and refer to a specific set of peptides in bacteria’s and fungi’s secondary metabolism. With the actual purpose of providing advantages within their respective ecological niche, the bioactivity of the structurally highly diverse products ranges from, e.g., antibiotic (e.g., vancomycin) to immunosuppressive (e.g., cyclosporin A) to cytostatic (e.g., echinomycin or thiocoralin) activity. An NRPS module consists of at least three core domains that are essential for the incorporation of specific substrates with the 'multiple carrier thiotemplate mechanism' into a growing peptide chain: an adenylation (A) domain selects and activates a cognate amino acid; a thiolation (T) domain shuffles the activated amino acid and the growing peptide chain, which are attached at its post-translationally 4ʹ-phosphopantetheine (4'-PPant) group, between the active sites; a condensation (C) domain links the upstream and downstream substrates. NRPS synthesis is finished with the transfer of the assembled peptide to the C-terminal chain-terminating domain. Accordingly, the intermediate is either released by hydrolysis as a linear peptide chain or by an intramolecular nucleophilic attack as a cyclic peptide. The NRPS’s modular character seems to imply straightforward engineering to take advantage of their features but appears to be more challenging. Since the pioneering NRPS engineering approaches focused on the reprogramming and replacement of A domains, several working groups developed advanced methods to perform a complete replacement of subdomains or single or multiple catalytic domains. The first part of this work focusses parts of the publication with the title 'De novo design and engineering of non-ribosomal peptide synthetases', which follows up assembly line engineering with the development of a new guideline. Thereby, the pseudodimeric V-shaped structure of the C domain is exploited to separate the N-terminal (CDSub) and C-terminal (CASub) subdomains alongside a four-AA-long linker. This results in the creation of self-contained, catalytically active CASub-A-T-CDSub (XUC) building blocks. As an advantage over the previous XU concept, the characteristics (substrate- and stereoselectivity) assigned to the C domain subunits are likewise exchanged, and thus, no longer represent a barrier. Furthermore, with the XUC concept, no important interdomain interfaces are disrupted during the catalytic cycle of NRPS, allow to expect much higher production titers. Moreover, the XUC concept shows a more flexible application within its genus origin of building blocks to create peptide libraries. Additionally, with this concept only 80 different XUC building blocks are needed to cover the entire proteinogenic amino acid spectrum. The second part of this work addresses the influence of the C domain on activity and specificity of A domains. In a comprehensive analysis, a clear influence of different C domains on the in vitro activation rate and the in vivo substrate spectrum could be observed. Further in situ and in silico characterizations indicate that these influences are neither the result of the respective A domains promiscuity nor the C domain’s proofreading, but due to an 'extended gatekeeping' function of the C domain. This novel term of an 'extended gatekeeping' function describes the very nature of interfaces that C domains can form with an A domain of interest. Therefore, the C-A interface is assumed to have a more significant contribution to a selectivity filter function. The third part of this work combines the NRPS engineering with phylogenetic/evolutionary perspectives. At first, the C-A interface could be precisely defined and further identified to encode equivalent information corresponding to the complete C-A didomain. Moreover, the comparison of NRPSs topology reveals hints for a co-evolutionary relatedness of the C-A didomain and could be shown to reassemble even after separation. In this regard, based on a designed CAopt.py algorithm, the reassembling-compatibility of hybrid interfaces could be determined by scoring of the co-expressed NRPS hybrids. This algorithm also enables the randomization of the interface sequences, thus, leading to the identification of more functional interface variant, which cause significantly higher peptide production and could even be applied to other native and hybrid interfaces.

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Neue Strukturdaten geben Einblick in die Bewegungen von Transportproteinen in der nicht‐ribosomalen Peptidsynthese

Cited by 6 publications

References 53 publications

Cascades in Compartments: En Route to Machine‐Assisted Biotechnology

Cascades in Compartments: En Route to Machine‐Assisted Biotechnology

Kaskaden in Kompartimenten: auf dem Weg zu maschinengestützter Biotechnologie

Non-ribosomal peptide synthetase engineering focusing on the condensation domain and the condensation/adenylation domain interface

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